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In this issue of Circulation Research, Tiruppathi and collaborators present a second report on the phenotypic changes that develop in mice lacking the TRPC4 channels developed by the Flockerzi group at the Institute for Pharmacology and Toxicology of the University of Saarland, Germany.1 This and the earlier report2 show unequivocally that TRPC channels and the Ca2+ whose entry they mediate are central players in cellular and organismic homeostasis.

Increases in cytosolic Ca2+ drive a myriad of cellular responses to extracellular stimuli, either alone or in conjunction with other signaling pathways. Examples are skeletal and smooth muscle contraction, neuronal and endocrine secretions, and activation of B and T lymphocytes, of neutrophil chemotaxis, of endothelial cell NO production, and of platelet aggregation. With the exception of skeletal muscle contraction, which is mediated exclusively by Ca2+ released from the sarcoplasmic reticulum in response to sarcolemmal depolarization, all other cellular responses enabled by Ca2+ depend on Ca2+ entering from the extracellular space through Ca2+-permeable ion channels. True to this adage, cardiac and smooth muscle contraction and neuro-, endo-, and exocrine secretions fail upon removal of extracellular Ca2+. Cells are negatively affected by sustained high cytosolic Ca2+ ([Ca2+]i) and expend significant amounts of energy to keep [Ca2+]i at low, ≈100 nmol/L levels—104 times lower than present in the extracellular milieu. Entry of Ca2+ into cells is thus carefully gated. The gating is performed by just three classes of ion channels: (1) voltage-gated Ca2+ channels, structural relatives of voltage-gated sodium channels, expressed in neuronal, endocrine, and muscle cells; (2) ligand-gated ion channels, including cyclic nucleotide- and excitatory amino acid-activated ion channels, expressed in various sensory and neuronal cells; and (3) an ubiquitous set of voltage-independent, Ca2+-permeable cation channels activated for the most part by maneuvers that include activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3). The molecular nature of these channels is just now coming to light.

Agents that lead to IP3 formation are those that act through receptors that are coupled to intracellular responses by either PLCß activated by G proteins of the Gq and Gi/o classes, or PLCγ activated by tyrosine kinases, thus reflecting the distinct responsiveness of these two types of PLC. PLCβs respond to Gq-derived GTP-Gαq and to Gi/o-derived Gβγ; PLCγs are activated by tyrosine phosphorylation. Stimulation of dedifferentiated fibroblast cell lines that lack voltage- and ligand-gated ion channels, in the presence of extracellular Ca2+, with an agonist that acts through a Gq-coupled receptor, typically shows an initial, rapid (within seconds) rise of [Ca2+]i to peak levels that can be as high as 100- to a 1000-fold basal, followed by a decline to a plateau level that averages between 2- and 4-fold the prestimulation level and persists until the stimulating agent is removed. The same experiment without extracellular Ca2+ shows the initial peak to be little affected but the plateau to be abolished. Readmission of Ca2+ to the medium after [Ca2+]i has returned to baseline—agonist continuously present—now shows an immediate influx of Ca2+ and establishment of the plateau phase of elevated [Ca2+]i that persists until the causative agonist is removed. With small variations, this type of experiment has been repeated with the same outcome by researchers across the world with almost all cells that have been isolated and tested for their [Ca2+]i changes in response to agonists, chemokines, cytokines, neurotrophic peptides, etc. Cells with this type of [Ca2+]i response pattern are not only of the nonexcitable type but also include excitable cells such as neurons and myocytes, visualized after blocking their voltage- and/or ligand-gated channels. This underscores the ubiquitous nature of the [Ca2+]i response to maneuvers that elevate IP3 levels in cells, and by extension, the ubiquitous nature of the Ca2+ entry pathway activated in conjunction with increases in cytosolic IP3.

What are the molecular components of this Ca2+ entry pathway? The hunt for channels through which the Ca2+ enters began ≈10 years ago with clues coming from studies with mammalian cells and insect photoreceptor cells. Mammalian cells were found to activate a similar Ca2+ entry pathway without prior stimulation of IP3 formation by simply doing what IP3 does, ie, by depleting the internal stores with the drug thapsigargin (TG). IP3 does this by binding to its intracellular receptor, the IP3R, which is a Ca2+ release channel located in the endoplasmic reticulum membrane that delimits the internal store. TG instead acts by inhibiting sarcoplasmic-endoplasmic reticulum ATPases (SERCAs), the Ca2+ pumps responsible for transporting Ca2+ from cytosol to the store.3 Ca2+ entry promoted by mere store depletion without overt activation of a PLC system was referred to as capacitative Ca2+ entry, for it eventually restored the capacity of the store to release Ca2+.4 Experimental maneuvers could now differentiate between agonist and IP3-mediated Ca2+ entry from mere store depletion-induced Ca2+ entry.

Although susceptible to blockade by nonspecific agents that block all Ca2+-permeable ion channels, such as the lanthanides, curiously—or strangely—this universal form of Ca2+ influx knows of no specific inhibitors. Electrophysiological characterization of the channel(s) through which Ca2+ enters cells after store depletion was elusive, until in 1992, Hoth and Penner5 characterized a store depletion-activated current in mast cells. Noise analysis indicated that single-channel currents had to be in the sub-picosiemen (pS) range. Ion permeation studies showed the channel to be exquisitely selective for Ca2+ over other divalent cations, including Mg2+. The term ICRAC for Ca2+ release-activated current was coined. In 1993, Penner’s group6 reported the existence in the same cells of receptor-activated nonspecific (Ca2+-permeable) cation channels with much larger single-channel conductances than those responsible for ICRAC, 50 pS, and thus laid the foundation for the fact that Ca2+ entry activated by the agonist-PLC-IP3 pathway, which includes store-activated capacitative Ca2+ entry channels, were likely to be heterogeneous. Numerous other examples confirmed this assumption.

TRP channels entered the scene at about the same time, when in 1992 Hardie and Minke7 reported that a light-activated Ca2+ current responsible for the sustained phase of the fly’s compound eye electroretinogram was absent in a Drosophila mutant termed transient receptor potential, TRP. In invertebrates, unlike in vertebrates, light activation of rhodopsin, instead of hyperpolarizing the photoreceptor cell, causes its depolarization through a pathway formed of rhodopsin, a Gq-like G protein, and PLC, akin to the one that elicits in mammalian cells the IP3- and store depletion-activated Ca2+ entry. Interestingly, the trp gene product predicted a structure with some characteristics of an ion channel with limited sequence homology to voltage-gated Na+ and Ca2+ channels.8 Could it be that mammalian homologues existed that are responsible for store depletion- and/or agonist-activated Ca2+ entries? By 1996, six mammalian TRP homologues (now referred to as TRPC channels) had been identified.9 Full-length cDNAs were available for several of them by 1997, including TRPC4, originally referred to as Trp4 and CCE1.10

Absence of cells lacking capacitative and/or PLC-IP3-activated Ca2+ entry made it difficult to establish physiological role(s) for the proteins encoded in TRPC cDNAs. Seven TRPC genes were eventually identified. Importantly, expression of none recapitulated ICRAC in terms of ion selectivity. Although TRPC1, TRPC2, and TRPC4, when expressed in model cells, were susceptible to activation by mere store depletion (TG treatment), channels appearing in TRPC3- and TRPC6-transfected cells seem insensitive to store depletion and require a receptor-PLC-IP3-IP3R pathway for their activation.

Formation of hybrid TRPC channels composed of more than one TRPC is at present the best explanation for the elusiveness of ICRAC properties of expressed TRPC cDNAs and the relative paucity of data from normal cells that predict the electrophysiological characteristics of the channels that appear upon transfection of TRPC cDNAs. In their quest for unequivocal roles, matters turned out still worse for TRPCs, the close homologues to Drosophila TRP, for they have been found to form part of a superfamily of TRP-related proteins. TRP-related proteins include the vanilloid and vanilloid-related receptors (VR1 and VRL1) and the cold and menthol receptor (CMR1), responsible for heat and cold sensing, the presumed anti-oncogene melastatin-1 (MLSN1), the polycystic kidney disease 2 gene product (PKD2), the epithelial calcium channels ECaC1 and ECaC2 (also CaT2 and CaT1), and channels with unrelated protein folds in their C-terminal domains such as an atypical kinase that interacts with PLC (TRP-PLIK) or a NuDix domain able to sense reactive oxygen species and ADP-ribose (see recent reviews11,12⇓; see nomenclature rules13).

Although not addressing specifically the question of subunit complexity of TRP channels—this may have to be solved by applying proteomics approaches—targeted inactivation studies are beginning to shed light on the functional significance of individual TRP channels. So, for example, an antibody approach showed an essential role of TRPC2 in sperm’s acrosome reaction triggered by oocyte zona pellucida protein,14 and ablation studies showed that TRPM7 (TRP-PLIK/LTRPC7) is essential for cell viability15 and that TRPC4 (Trp4/CCE1) contributes to endothelium-mediated vascular smooth muscle relaxation.2

Tiruppathi et al expand the studies of Freichel and collaborators with TRPC4−/− mice2 by studying properties of lung endothelial cells both in isolation and in situ.1 Thrombin, or a thrombin receptor-derived peptide with agonist activity, was used as a trigger of Gq activation to regulate endothelial cell functions such as Ca2+ entry, stress fiber formation, and cellular retraction. Further, in a model that assesses endothelial cell-dependent microvessel liquid permeability, they measured thrombin-induced permeability increases. In all instances, loss of TRPC4 correlates with a loss of endothelial cell responses, showing participation of TRPC4 in the response of endothelial cells to thrombin or its surrogate PAR1 receptor-derived agonist. Reduction of Ca2+ influx was expected from previous results with TRPC4−/− aortic endothelial cells,2 but the strict dependence on agonist-activated Ca2+ entry for stress fiber formation, and its function in retraction and relaxation of cell-cell interaction as seen in the microvascular permeability studies, was not.

It is noteworthy that impairment of thrombin’s responses was not always total. In aortic strips, endothelium-dependent Gq-activated relaxation was lost by only 50%.2 Likewise, endothelial cell retraction and microvasculature permeability increases were only reduced by ≈50% in TRPC4−/− mice. On the other hand, stress fiber formation was fully eliminated, and Ca2+ influx assessed by the Ca2+ readdition assay was reduced by 80% to 90%. These results very likely indicate that other TRPCs or one or more of the TRP-related molecules—some known, others just suspected of having the ability to form ion channels—may cover for the loss of TRPC4 in endothelial cell function. It is for future research to answer these questions. In the meantime, it is clear that TRPC4, originally called CCE1, is indeed a critical component of capacitative Ca2+ influx, thus validating the efforts of many in the field who, like me, betted on TRPCs being important in agonist and capacitative Ca2+ entry.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.